Natural herbal extract roles and mechanisms in treating cerebral ischemia: A systematic review

Background Stroke has been the focus of medical research due to its serious consequences and sequelae. Among the tens of millions of new stroke patients every year, cerebral ischemia patients account for the vast majority. While cerebral ischemia drug research and development is still ongoing, most drugs are terminated at preclinical stages due to their unacceptable toxic side effects. In recent years, natural herbs have received considerable attention in the pharmaceutical research and development field due to their low toxicity levels. Numerous studies have shown that natural herbs exert actions that cannot be ignored when treating cerebral ischemia. Methods We reviewed and summarized the therapeutic effects and mechanisms of different natural herbal extracts on cerebral ischemia to promote their application in this field. We used keywords such as “natural herbal extract,” “herbal medicine,” “Chinese herbal medicine” and “cerebral ischemia” to comprehensively search PubMed, ScienceDirect, ScienceNet, CNKI, and Wanfang databases, after which we conducted a detailed screening and review strategy. Results We included 120 high-quality studies up to 10 January 2024. Natural herbal extracts had significant roles in cerebral ischemia treatments via several molecular mechanisms, such as improving regional blood flow disorders, protecting the blood-brain barrier, and inhibiting neuronal apoptosis, oxidative stress and inflammatory responses. Conclusion Natural herbal extracts are represented by low toxicity and high curative effects, and will become indispensable therapeutic options in the cerebral ischemia treatment field.


Introduction
Cerebral ischemia (CI) is a complex disease in clinical medicine.To put it simply, due to various reasons, blood in brain tissue cannot support normal metabolism and function, with subsequent symptoms collectively referred to as CI.Worldwide, morbidity and mortality rates due to CI are very high.The disease is characterized by several etiologies, changeable conditions, and serious consequences, which exert extremely heavy burdens on patients and their families.The National Institutes of Health Stroke Scale is commonly used to assess neurological damage in patients with clinical ischemia.Even if a patient avoids death, most will experience severe neurological dysfunction.Currently, the early treatment of patients with CI mainly occurs via the rapid restoration of cerebral blood flow perfusion, however, this restoration increases oxidative stress and inflammatory responses in ischemic tissue, leading to reperfusion injury.One factor that determines the severity of a patient's condition is ischemia duration, which is a very important determinant when selecting treatment options for patients with acute cerebral ischemia (ACI) (Powers, 2020).Another problem that cannot be ignored is that the recombinant tissue plasminogen activator (rt-PA) drug is currently approved by the U.S. Food and Drug Administration for stroke patients, but its treatment window is very narrow and it has very serious side effects (e.g., cerebral hemorrhaging) (Dhamija and Donnan, 2007).Thus, a lack of drugs is an urgent issue for CI treatment.To remedy this, promoting lowtoxicity and high-efficiency drug research and development can alleviate CI patient suffering.Our work is based on this purpose and motivation.
Herbal medicines are gifts from nature, and have helped humans solve medical problems that have plagued humankind for centuries.For example, artemisinin extracted from Artemisia annua L. helps alleviate malaria (Talman et al., 2019).Methanol extracts from Allium turcicum Özhatay and Cowley exert significant anticancer, antioxidant, and antimicrobial activities (łpek et al., 2024).These natural herbal extracts (NHEs) have active roles in many different fields, for example, a Chenopodium quinoa Willd.seed extract restores photosystem II damage caused by toxic metal salts (Ganieva et al., 2023).Also, Pistacia atlantica Desf.extracts effectively inhibit Fusarium oxysporum f. sp.albedinis to rescue infected date palms (Fatiha et al., 2023).NHEs have unique structures and GRAPHICAL ABSTRACT properties, and are roughly divided into alkaloids, flavonoids, polysaccharides, glycosides, organic acids, and volatile oils.

Alkaloids
Alkaloids are nitrogen-containing organic compounds mainly found in plants, which have similar chemical properties to alkalis.One common property is that they all contain nitrogen as part of their chemical structure, but not all organic compounds containing nitrogen are alkaloids.Thanks to natural herb research and exploration, nearly 10,000 alkaloids have been discovered and collected.Alkaloids are subdivided into more than 60 types, for example, Leonurus japonicus Houtt.total alkaloid (LHA) is an organic amine in alkaloids.LHA and kukoamine A (KuA) are common alkaloids.LHA helps protect the blood-brain barrier (BBB) and inhibits inflammatory reactions and apoptosis in ischemia-reperfusion (IR) injury (Zhang Q.-Y. et al., 2017;Li Y. et al., 2021).KuA impacts CI injury by alleviating brain edema and inhibiting oxidative stress and apoptosis (Liu et al., 2017).

Flavonoids
Flavonoids are one of the most widespread organic compounds in nature; they exist in almost all green plants, especially higher plants.In the selected studies in this review, many have investigated flavonoid NHEs, such as emodin, scutellarin, and icariin (ICA).From our research, emodin enhances cell viability and inhibits oxidative stress radicalization to alleviate IR injury (Wang et al., 2007;Leung et al., 2020); scutellarin improves neurological dysfunction in rats during IR injury by inhibiting apoptosis, focal death, and necrosis (Wang C. et al., 2023); and ICA inhibits apoptosis and protects neuronal dendrites during chronic cerebral ischemia (CCI) to improve cognitive impairment (Li W.-X. et al., 2015).

Polysaccharides
Polysaccharides are macromolecules composed of at least 10 monosaccharides, and have important roles maintaining normal life activities.Polysaccharides are divided into plant, animal, and fungal polysaccharides according to extraction sources.Plant polysaccharides include ganoderma polysaccharides, lentinan, ginseng polysaccharides, and other polysaccharides beneficial to humans.Previous studies have reported that some polysaccharides may have anti-tumor effects (Zhang et al., 2021).

Glycosides
Glycosides have high medicinal value, which not only enhance immunity and antiviral effects, but also inhibit oxidative stress and enhance metabolic function in cells.Astragaloside IV (ASIV), ginsenoside, and notoginsenoside are well-known representative glycosides, especially ASIV.As an Astragalus membranaceus (Fisch.)Bunge extract, ASIV inhibits inflammatory reactions, promotes neurogenesis and angiogenesis, promotes neurotrophic factor expression, protects the BBB, and significantly improves neurological dysfunction caused by IR injury (Li et al., 2013;Li L. et al., 2021;Li S. et al., 2021;Shi et al., 2021).Ginsenoside has protective roles in injury caused by ACI, CCI, and IR.It not only inhibits apoptosis, increases angiogenesis, and improves local blood flow disorders, but also protects the BBB and improves cognitive dysfunction caused by CI (Zhou et al., 2014;Yang et al., 2016;Wang S. et al., 2017;Wan et al., 2017;Zhang et al., 2019;Zhang C. et al., 2020).Notoginsenoside resists injury caused by ACI and IR by alleviating brain edema, enhancing cell viability, protecting the BBB, and inhibiting apoptosis (Tu et al., 2018;Liu B. et al., 2021;Gao et al., 2022;Liu et al., 2022).

Volatile oils
Volatile oils mainly come from aromatic traditional Chinese medicine, with many fragrant plants more or less containing these compounds.The group includes terpenoids and aromatic compounds and also their oxygen-containing derivatives such as alcohols, aldehydes, ketones, phenols, ethers, and lipids.Additionally, the group includes some nitrogen-and sulfurcontaining compounds.The most common NHEs are ginkgolide and ligustilide (LIG).Seven ginkgolide species have been found: A, B, C, M, J, K, and L. Ginkgolide B (GB) exerts the greatest effects, so many pharmacological studies have focused on this compound.GB protects the BBB and improves mitochondrial respiratory function against ACI injury (Li et al., 2007).LIG alleviates neurological deficits and inhibits apoptosis, astrocyte activation and proliferation, and oxidative stress responses in a CCI model (Feng et al., 2012;Peng D. et al., 2022).

Search strategy
To comprehensively and systematically conduct literature retrieval and data extraction, we preliminarily searched and screened all studies from PubMed, ScienceDirect, Web of Science, CNKI, and WANFANG databases before 10 January 2024 in strict accordance with Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) guidelines.The retrieval keywords are as follows: 1) natural herbal extracts, 2) Chinese herbal medicines, 3) herbal medicines, and 4) cerebral ischemia.Constructed retrieval expressions are (natural herbal extracts OR Chinese herbal medicines OR herbal medicines) AND cerebral ischemia.After retrieving pertinent studies (n = 7,018), we preliminarily screened (using titles, keywords, and abstracts) and comprehensively reviewed these studies.Finally, 120 studies were selected for review (Figure 1).

Inclusion and exclusion criteria
In view of the therapeutic effects and mechanisms of NHEs toward CI, and the large number of studies, we formulated the following inclusion and exclusion criteria.Inclusion criteria; 1) A cerebral ischemia model and 2) a control group are included; 3) At least one experimental group used NHEs as an intervention; and 4) Research data are published in high impact journals.Exclusion criteria; 1) Reviews or books; and 2) Studies on other diseases and compounds.

Data extraction and treatment evaluation
The authors independently extracted and summarized information from selected studies, solved any issues via discussion, and finally summarized the information, including; 1) NHEs; 2) The source and voucher numbers of the herbal medicine; 3) Extraction methods 4) Extraction parts and solvents; 5) Toxicity and side effects; 6) Related diseases in the study; 7) Establishing in vitro or in vivo models; 8) Animal or cell models; 9) Dose and time of treatment; 10) Main biological effects; 11) Mechanism of action; 12) Year of publication and first author; and 13) Positive controls.

Risk of bias
After discussions, the authors referred to the previous literature (Dong et al., 2023) to establish an evaluation scale of bias risk for selected studies.The following questions were posed; 1) Had the study passed peer review; 2) Were randomness principles followed when grouping models; 3) Were blinding methods applied during drug interventions and data collection; 4) Were sample sizes statistically calculated before model establishment; 5) Were animal welfare laws and regulations strictly observed in the research process; and 6) Were potential conflicts of interest between authors declared?

Results
From our strategy, 7,018 studies were retrieved, including 2032 Pubmed results, 1,242 Web of Science results, 3,578 ScienceDirect results, 124 CNKI results, and 42 WANFANG results.After preliminary screening and applying inclusion and exclusion, 120 studies were finally selected (Figure 1).Among these, 21 were related to ACI, 28 to CCI, and 71 to IR.In order to avoid disease subtype differences which may have affected our study results, we separately summarized the therapeutic effects of NHEs for ACI, CCI, and IR.

ACI
In the 21 ACI-related studies were 19 in vivo and nine in vitro studies, from which we summarized the characteristics of each study (Table 1).To ensure reliability, we summarized in vivo and in vitro study characteristics, separately.In the 19 in vivo studies, NHEs mitigated ACI-induced damage via different modes of action (Table 2).In nine in vitro studies, damaged cell models achieved varying degrees of remission after a NHE intervention (Table 3).

CCI
We included 28 studies on CCI, including 28 in vivo and four in vitro studies.We summarized CCI-related research characteristics (Table 4).NHEs alleviated damage caused by CCI and improved cognitive dysfunction caused by ischemia (

Cerebral IR
Of the 71 cerebral IR-related studies, 68 were in vivo and 31 were in vitro based.A summary of study characteristics is shown (Table 7), and then we describe the studies in vivo and in vitro, separately.Studies showed that NHEs had therapeutic roles in in vivo brain IR models (Supplementary Table S1).NHEs also reduced IR damage in in vitro models (Table 8).

Quality evaluation of selected studies
Using our bias risk assessment scale, bias risk assessments were conducted on the 120 studies.All were peer-reviewed publications and they strictly complied with animal welfare regulations, which meant that all studies had at least two points (total score = 6 points).Additionally, 120 studies followed randomization principles (84.17%), 39 adopted blind methods (32.5%), 88 declared conflicts of interest among authors (73.33%), and only one study statistically calculated the sample size (0.83%) (Figure 2).Perhaps some researchers had calculated sample sizes before their studies, but this was not stated.After evaluations, bias risk scores for studies were in the 2-6 range: six studies scored 2 (5%), 29 scored 3 (24.17%),56 scored 4 (46.67%),28 scored 5 (23.33%), and one scored 6 (0.83%).Approximately half (46.67%) received four points, which proved that study quality was high.Bias risk evaluations for studies are shown (Table 7).

Toxicity
Unfortunately, many studies failed to provide NHE-related toxicity information.While drug toxicity studies are usually conducted at pre-experimental stages, researchers must articulate this.To complement the required NHE toxicity reports for this review, we performed additional NHE safety reviews by summarizing the toxicity reports in selected studies (Supplementary Table S2).However, not all NHEs have accompanying toxicity reports, which undoubtedly confirms a lack of safety studies in the natural herb research field.Reported that the LD50 (median lethal dose, the minimum amount of toxin required to kill half of an animal of a certain weight or age within a specified period of time) of SAA in mice (the dose required to kill half of cells/animal after a specific trial duration) was 1,161.2mg/kg (Yang M.-Y. et al., 2022).G. biloba extracts may be carcinogenic and caution is recommended for their long-term use (Mei et al., 2017).ASIV is not toxic during maternal and embryonic development, but may inhibit fertility in female rats, suggesting its non-use in perinatal periods (Xuying et al., 2010).Luo et al. reported that emodin reduces and inhibits human sperm motility, suggesting some reproductive toxicity in these cells (Luo et al., 2015).LHA did not generate significant adverse reactions in toxicity tests in multiple experimental animals (Zhu et al., 2018).
The main component of an ethanolic extract from Erythrina velutina Willd.(EEEV) is gallic acid, with in vivo studies showing that gallic acid at 210 mg/kg exerted no toxic effects in mice (Li et al., 2019).Ginkgolide A and ginkgolide B reduce mouse blastocyst viability and cause embryonic retardation in mice, leading to embryo death, suggesting caution when using these reagents during pregnancy (Mei et al., 2017).The main component of supercritical CO2 extracts from DanShen (SCED) is tanshinone IIA (TSA); it was found that at high TSA concentrations (25M), zebrafish embryo models exhibited severe growth inhibition, developmental malformations, and cardiotoxicity (Wang T. et al., 2017).Usually there are no obvious side effects when Panax notoginseng is supplemented to patients, but due to its estrogen effects, some patients have reported vaginal bleeding and distending breast pain.Those patients taking high P. notoginseng doses (>2.5 g/ day) have central nervous system damaging effects such as insomnia, tachyarrhythmia, hypertension, and tension (Mancuso and Santangelo, 2017).In vivo betulinic acid studies showed no signs of systemic toxicity (Liu C. et al., 2019).Scutellarin has the lowest toxicity in rodents, and can even be said to be non-toxic.Quercetin (Que) toxicity is low; the organ weights and histopathology of rats treated with 400 mg/kg/d Que for 410 consecutive days showed no significant changes.Ginsenoside Rd, ginsenoside Rb1, and notoginsenoside promote cancer cell apoptosis and have significant effects in cancer treatment.Notoginsenoside R1 also inhibits cell proliferation, migration, invasion and angiogenesis, and promotes cell apoptosis at 150 µM.Shikonin is considered safe, but may cause skin allergies at very low doses.Luteolin exerts cytotoxicity at 5 μM and 10 µM doses, and its safety must be further evaluated in animal models and clinical trials.Hydroxysafflor yellow A is sensitive to interactions between herbs and drugs, resulting in no therapeutic effects at certain doses (Guo et al., 2023).Galangin has an IC50 (half-inhibitory concentration, the concentration at which a biological process or activity is inhibited by 50%) value of 275.48 μM in V79 cells, and does not produce genotoxic effects at all concentrations (Bacanlı et al., 2017).Similarly, echinocystic acid (EA) exerts no cytotoxic effects under any conditions in cell viability assays (Joh et al., 2012).Glycyrrhizin is moderately toxic and should be used with caution during pregnancy.It also has selective cytotoxic effects toward cancer cells, and its most important side effects are secondary diseases induced by hypertension and hypokalemia (Nazari et al., 2017).After the maternal application of 1.0 mg/kg ASIV for 28 consecutive days, ASIV delays development in young rats and should be used with caution in perinatal women (Zhang J. et al., 2020).Studies report serious adverse reactions to matrine, the most serious being hepatotoxicity, neurotoxicity, and reproductive toxicity (Wang X. et al., 2023).Acute and subacute toxicity studies report that breviscapine is a safe drug with a potential for widespread use in clinical settings (Wu et al., 2021).
Icariin has an IC50 of 20 μM in HeLa cells, and its toxic effects in normal cells are relatively negligible (Huang et al., 2019).

Discussion
We comprehensively summarized the molecular mechanisms underlying CI treatment by NHEs (Figure 3).As mentioned, NHE therapeutic effects toward CI are roughly divided in two ways: one  growth factor A (VEGFA), and intercellular adhesion molecule 1 (ICAM1), which means that SAA not only inhibits apoptosis, inflammation, and oxidative stress, and protects the BBB, but also promotes vascular proliferation and recovery (Jiang et al., 2011;Chien et al., 2016;Song et al., 2019;Liu C. et al., 2021;Ling et al., 2021).Additionally, some key factors involved in multiple pathways, such as VEGF, are activated by multiple NHEs.The discovery of VEGF has completely changed our understanding of blood vessel production during development and physiological homeostasis.The biological effects mediated by VEGF are mainly due to its impact on vascular permeability and new blood vessel generation.VEGF has important relationships with tumor growth and metastasis, hypertensive retinopathy, and other pathological conditions (Apte et al., 2019).T-VA is extracted from Ligusticum sinense (Li G. et al., 2015).Several studies report that T-VA, ginsenoside Rb1 (GSRb1), L-borneol, and DL-n-butylphthalide (DL-NBP) can also play a role through VEGF (Li G. et al., 2015;Zhu et al., 2018;Ma et al., 2021;Wang et al., 2020).CI is a severe nerve injury caused by interrupted cerebral blood flow.The molecular mechanisms underpinning its pathological processes are extremely complex and cannot be fully explained at present.CI involves amino acid excitation, injury oxidative stress, inflammatory responses, BBB injury, mitochondrial dysfunction, cell necrosis, and apoptosis.Many studies report that key molecules are involved in these CI-mediated processes, such as hypoxiainducible factor alpha (HIF-1α), VEGF, brain-derived neurotrophic factor (BDNF), protein kinase B (Akt), matrix metalloproteinases (MMPs), c-Jun N-terminal kinase (JNK), B-cell lymphoma-2-associated X (Bax), Caspase-3/9, mitogenactivated protein kinases (MAPKs) and nuclear factor kappa-B (NF-κB) (Wang et al., 2020;Li et al., 2022;Liu et al., 2023;Li et al., 2017;Chen H.-S. et al., 2018;Hu et al., 2020;Peng T. et al., 2022;Xu et al., 2021).
Blood flow disturbance is the most fundamental issue in CI, and appears to initiate several pathological conditions, such as BBB damage, mitochondrial dysfunction, cell necrosis, and apoptosis.To reduce the severity and prognosis of stroke onset, doctors must rapidly conduct clinical interventions such as intravenous thrombolysis and surgical thrombectomy to unblock cerebral blood vessels.Anticoagulant and antiplatelet therapies are recommended for patients without contraindications, but their harsh conditions of use and severe side effects have prompted scientists to explore better treatments.Thromboxane A2 (TXA2) is a potent vasoconstrictor and the main cyclooxygenase (COX) product of arachidonic acid (AA).The functional importance of this eicosanoid in acute coronary ischemic syndrome has been demonstrated as it activates platelets (Reilly and Fitzgerald, 1993).Fei et al. extracted natural compounds with TSA as the main component from S. miltiorrhiza Bunge (DanShen); this SCED inhibits platelet aggregation and improves regional blood flow disorders by inhibiting TXA2 activation (Fei et al., 2017).Although the specific molecular mechanisms have not been clarified, and to determine NHE biological effects on blood flow disorders, several studies have used laser speckle imaging to show that P. notoginseng saponins (PNS), GSRb1, L-borneol, and galangin can improve regional blood flow disorders after stroke (Li S. et al., 2012;Wang S. et al., 2017;Gao et al., 2022;Xie et al., 2023).Quality evaluation chart.Amino acid excitotoxicity is due to the abnormal accumulation of some excitatory amino acids (such as glutamate) outside neurons after ischemia.Glutamate accumulation leads to sustained Ca 2+ channel and N-methyl-D-aspartic acid receptor (NMDAR) activation on neuronal synaptic membranes.NMDAR is an ion channel regulated by glutamate on cell membranes.After glutamate activation, high Ca 2+ levels are transported into membranes.Ca 2+ accumulates in the cytoplasm and mitochondria, resulting in Ca 2+ overload.This alteration affects many biological processes, such as calpain activation, oxidative stress responses, and mitochondrial damage, and also protease, kinase, phosphatase, and other enzyme activities, leading to cell death.As a major transporter of excitatory amino acids, glutamate transporter 1 (GLT-1) is mainly distributed in astrocytes.Usually, GLT-1 mediates glutamate uptake by glial cells to maintain extracellular glutamate concentrations.GLT-1 function is impaired during CI, resulting in high intersynaptic glutamate accumulation (Rao et al., 2001).GSRb1 and emodin reportedly activate GLT-1 receptors on astrocyte membranes, transferring glutamate into astrocytes to reduce its abnormal accumulation outside neurons (Wang S. et al., 2017;Leung et al., 2020).GSRb1 also has the same effects as ginsenoside Rd (GSRd) in inhibiting NMDAR expression (Zhang C. et al., 2020).Glutamine synthetase (GS) catalyzes glutamate conversion to glutamine in vivo and has important roles regulating glutamate levels.KRGP is an active substance extracted from Korean red ginseng (Liu L. et al., 2019).Liu et al. found that GS expression levels are significantly elevated after CI in KRGP-pretreated mice, while GS expression levels are not changed much in nuclear factor erythroid 2-related factor 2 (Nrf2) gene deletion mice, suggesting that the Nrf2 pathway has important roles in glutamate homeostasis after CI, and that KRGP may reduce amino acid excitation damage caused by CI via Nrf2 signaling (Liu L. et al., 2019).
Oxidative stress is a common phenomenon in hypoxic cells.Mitochondria are essential organelles which maintain energy homeostasis in cells.The state and function of mitochondria undergo significant changes during hypoxia, leading to increased intracellular reactive oxygen species (ROS) levels, which severely damage cells and brain tissue.Oxidative stress products directly attack biomacromolecules (amino acids and nucleic acids) to induce apoptosis and increase BBB permeability.Nrf2 has antioxidant and anti-inflammatory effects that activate heme oxygenase-1 (HO-1) after oxidative stress-inducer (e.g., inflammatory chemokines/ cytokines) activation or tissue damage (Hassanein et al., 2023).HO-1 is an inducible homolog with antioxidant properties and has important roles regulating oxidative stress, with elevated HO-1 levels detected in almost all oxidatively stressed cells.catalpol, KRGP, CZ-7, AA, and ruscogenin (Rus) increase HO-1 expression by stimulating Nrf2 (Liu L. et al., 2019;Liu D.-D. et al., 2019;Wang et al., 2022;Zhang S. et al., 2023;Kim et al., 2023).Additionally, by detecting mitochondrial energy metabolismrelated genes (Atp12a and Atp6v1g3), Liu et al. found that notoginsenoside R1 (NGR1) mitigates mitochondrial energy metabolism abnormalities (Liu et al., 2022).Three Nei-like DNA glycosylases exist in mammalian cells, which protect DNA by removing oxidative bases.GSRd protects neurons by activating Nei-like DNA glycosylase 1/3 (NEIL1/3) to promote DNA hydrolysis of oxidative stress-induced product damage (Yang et al., 2016).
Inflammatory responses are self-defense mechanisms; they are stimulated by endogenous and exogenous inflammatory factors and are closely related to different diseases.Neuroinflammation occurs at almost all stages of ischemic stroke and is caused by damage-associated molecular pattern release by damaged/dead cells.These patterns, including adenosine, heat shock proteins, and interleukin 33 (IL-33) are recognized by corresponding immune cells which trigger multiple downstream signaling pathways (Qin et al., 2022).Additionally, these patterns stimulate inflammation-related cytokine, interferon or chemokine production, leading to increased adhesion molecule expression, helping white blood cells adhere to blood vessel surfaces, and promoting immune cell infiltration.Therefore, for patients with CI, early antiinflammatory treatment is an important method to reduce ischemic injury and improve prognosis.Pro-inflammatory cytokines induce chemokine secretion immediately after CI.Chemokine-chemokineligand2 (CCL2) and its receptors are involved in regulating inflammation in the ischemic state, and may be recruited to and adhere to cerebral vascular endothelial cells by immune cells.Signal transducer and activator of transcription 3 (STAT3) has positive regulatory effects on chemokines (such as CCL2) and acts as a key transcription factor during inflammation and immunity.Li et al. reports that ASIV inhibits CCL2 functions by inhibiting STAT3 expression and inhibiting NK cell infiltration (Li S. et al., 2021).JNK, TLR4, NF-κB, and MAPKs also have key roles in inflammatory signaling pathways via a vicious cycle between JNK and TLR4 (Cheng et al., 2021a).TLRs are expressed on cell surfaces and in intracellular spaces, and regulate the state and function of many immune cells.Fructus extracts, procyanidins, and SAA inhibit TLR4 expression (Lee et al., 2015;Yang et al., 2020;Ling et al., 2021), while Alpinia oxyphylla Miq.(YZR) extract and EA inhibit JNK activation (Yu et al., 2019;Cheng et al., 2021a).T-VA, silymarin, storax, and EK100 also suppress inflammatory responses by inhibiting NF-κB (Hou et al., 2010;Li G. et al., 2015;Wang et al., 2019;Zhou M. et al., 2021).The MAPK signaling pathway is activated shortly after ischemic injury onset.MAPK is composed of three major effectors, extracellular signal-related kinases (ERK1/2), JNK, and p38 MAPK.Among these, p38 MAPK regulates proinflammatory cytokine expression.The activation of MAPK/ ERK signaling and the stimulating effects of MMP expression can aggravate BBB injury in ischemic stroke and further enhance pro-inflammatory factor expression.Interestingly, we found that different NHEs have opposite effects on p38 MAPK, but all were protective against CI injury, which we speculate might be due to the activation of different factors downstream of p38 MAPK.L-borneol and Angelica sinensis (Oliv.)Diels (ASD) extracts activate p38 MAPK (Cheng et al., 2021b;Xie et al., 2023), while Honokiol and Scutellaria baicalensis Georgi (SB) extracts inhibit its function (Hwang et al., 2011;Chen et al., 2014).

Conclusion and prospects
With deepening research on NHEs, their mechanisms are becoming more complex and multifaceted.Complexity means that a single NHE, such as SAA, can simultaneously act on multiple molecular pathways.Multifaceted means that a NHE acting on the same key factor on a certain pathway may have different regulatory outcomes, such as L-borneol and Honokiol.In such cases, a deeper understanding of study conditions and results is required.Although too many pathways and factors are involved in these studies, key factors such as VEGF, BDNF, Akt, MMPs, JNK, Bax, Caspase-3/9, MAPKs, and NF-κB, may provide reference points for further research.Additionally, although CI pathogenesis is highly complex, the first and most important pathology is disturbed blood flow, which is why early CI treatments should rapidly restore blood supply.Therefore, more attention should be paid to NHEs (TSA or GSRb1) with antiplatelet or antithrombotic effects.For advanced CI treatments, selected studies should mainly focus on inhibiting neuroinflammation, inhibiting neuronal apoptosis, and protecting the BBB.This means that NHE treatment effects for CI are significant, and show how important neuroinflammation and neuronal apoptosis processes are in CI.In addition, the treatment of transient cerebral ischemia and permanent cerebral ischemia is also different.Transient cerebral ischemia is a sudden, transient cerebral vascular insufficiency, usually without brain tissue necrosis.The treatment of transient cerebral ischemia is mainly prevention, such as antiplatelet therapy.Permanent cerebral ischemia often has abnormal pathology such as thrombus, which leads to blood flow interruption and eventually brain cell death.If the vascular recanalization treatment cannot be carried out in time, serious sequelae will often be left.
Although strict review conditions were set, some flaws were identified in selected studies: 1) The elaboration of extraction protocols or NHE sources was not adequately detailed.Due to different NHE extraction methods, resultant NHEs may have different biological activities and effects, which makes the research data unreliable; 2) NHE toxicity was not adequately explored in selected studies.Even if some studies performed toxicity tests, they were in vitro and not in vivo; 3) Although considerable animal data were observed, it is uncertain if these NHEs can be eventually used in clinical practice; therefore, more pharmacological and pharmacological studies are required; and 4) NHE mechanisms were not fully explored.Most studies only explored one or several related factors, but did not examine complete NHE mechanisms.
Clinically observed NHE effects are the result of combined drug actions across a multitude of signaling pathways.Researchers are constantly exploring new treatment options to maximize treatment benefits while minimizing side effects.Traditional Chinese medicine efforts in this area are worthy of recognition, and the synergistic actions of multiple NHEs should be considered in future research.In addition, the reproducibility of drug efficacy is very critical, and it is an important factor affecting whether a drug can be transformed into clinical practice.In order to do this, we need to maintain a rigorous working attitude and record the experimental process in detail.We should strengthen our understanding of the various parts of the experiment and conduct sufficient pre-experiments.The factors affecting the transformation of medical achievements also include the lack of advanced medical equipment, insufficient attention to medical transformation, and lack of communication between the supply and demand sides.

TABLE 1
Summary of ACI study characteristics.

TABLE 2 (
Continued) NHE therapeutic effects and mechanisms in in vivo ACI models.

TABLE 4
Summary of CCI study characteristics.TABLE 5 NHE therapeutic effects and mechanisms in in vivo CCI models.

TABLE 5 (
Continued) NHE therapeutic effects and mechanisms in in vivo CCI models.

TABLE 6
NHE therapeutic effects and mechanisms in in vitro CCI models.
reduces damage, mainly by improving local blood flow disturbance, inhibiting oxidative stress, inflammatory responses and apoptosis, relieving cerebral edema, protecting the BBB, and inhibiting excitatory amino acid overexpression.The other way promotes injury recovery, mainly by promoting endothelial cell proliferation and migration, promoting neuron proliferation and differentiation, and promoting neurotrophic factor expression.NHEs may also act on molecules, such as SAA, via several pathways.SAA is a bioactive compound extracted from Salvia miltiorrhiza Bunge.Studies report that SAA has direct or indirect effects on toll-like receptor 2/4 (TLR2/4), phosphoinositide 3-kinase (PI3K), glycogen synthase kinase 3β (GSK3β), vascular endothelial

TABLE 7
Summary of IR study characteristics.

TABLE 8 (
Continued) NHE therapeutic effects and mechanisms in in vivo IR models.

TABLE 8 (
Continued) NHE therapeutic effects and mechanisms in in vivo IR models. .172944